Frequent problems and their resolutions by using thermal asymmetric interlaced PCR (TAIL-PCR) to clone genes in Arabidopsis T-DNA tagged mutants
Frequent problems and their resolutions by using thermal asymmetric interlaced PCR (TAIL-PCR) to clone genes in Arabidopsis T-DNA tagged mutants
复制标题
使用热不对称交错 PCR (TAIL-PCR) 克隆拟南芥 T-DNA 标记突变体基因时出现的常见问题及其解决方法
DOI：
10.1080/13102818.2014.998161
复制
发表时间：
2015
影响因子：
1.4
通讯作者：
Guo Guang-Qin
中科院分区：
文献类型：
--
作者：
Wu Lei;Di Dong-Wei;Zhang Dan;Song Bin;Luo Pan;Guo Guang-Qin
T-DNA插入突变是拟南芥功能基因组研究的有力工具。以往的研究已经发展出热不对称交错聚合酶链式反应(Tail-PCR)作为一种有效的策略来分离T-DNA标记突变体中与已知序列相邻的DNA序列。然而，当试图克隆T-DNA标记突变体中的侧翼序列时，会遇到一些问题。因此，有必要提高克隆诱变的效率。在这里，我们介绍了最常见的问题，并提供了一种改进的方法来提高Tail-PCR的效率。即使这样，也不是总能成功获得侧翼序列；在这种情况下，我们建议使用高通量测序来确定突变。
T-DNA insertional mutagenesis is a powerful tool inArabidopsisfunctional genomics research. Previous studies have developed thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR) as an efficient strategy in isolation of DNA sequences adjacent to known sequences in T-DNA tagged mutants. However, a number of problems are encountered when attempts are made to clone flanking sequences in T-DNA tagged mutants. Therefore, it is necessary to improve the efficiency of cloning mutagenesis. Here, we present the most frequent problems and provide an improved method to increase TAIL-PCR efficiency. Even then, it is not always possible to successfully obtain flanking sequences; in such cases, we recommend using high-throughput sequencing to determine the mutations.